Immunization to protect against Human Immunodeficiency Virus Type 1 (HIV-1) infections is a global priority. However, the efforts to develop an effective vaccine have been thus far unsuccessful in terms of protection of humans from acquisition of infection. The mechanism by which most viral vaccines protect against infection is through induction of antibodies that neutralize viral infectivity so that entry of the virus into cells of the vaccinated individual does not occur (Quinnan et al. (1997) Antiviral Agents and Human Viral Diseases, ed. Galasso, Whitley, and Merigan, Raven Press, pp. 791-834).
A major goal of efforts to develop a vaccine against HIV-1 is the induction of broadly cross-reactive neutralizing antibodies (Burton et al. (2004) Nat. Immunol. 5:233-236). Induction of antibodies that are highly potent and mediate neutralization of HIV-1 with broad cross-reactivity against epidemic strains has not yet been achieved.
The HIV-1 envelope glycoprotein complex (Env) is displayed on the surface of the virus and is the target of neutralizing antibodies. Two different proteins comprise the Env complex: gp120, the surface component, and gp41, the transmembrane component. Each Env complex is believed to consist of three copies of each of these two proteins in a trimer of heterodimers. The glycoproteins are initially produced during virus infection as a polyprotein, designated gp160. Cellular proteases cleave gp160 into the two subunits, gp120 and gp41, which remain non-covalently associated with each other in the Env complex.
The epitopes that are the targets of cross-reactive neutralizing antibodies are displayed on the surface of the trivalent complex, and they depend upon the quaternary structure of this complex.
Conformation-independent neutralization epitopes are located on both the surface, gp120, and transmembrane, gp41, components of the Env (Scanlan et al. (2002) J Virol 76, 7306-7321; Zwick et al. (2001) J Virol 75, 10892-10905; Wyatt et al. (1998) Nature 393, 705-711). There are also conformational epitopes associated with the heterotrimeric complex, some of which overlap receptor or co-receptor binding sites (Labrijn et al. (2003) J Virol 77, 10557-10565).
Among most HIV-1-infected patients, the degree of neutralizing antibody cross-reactivity that develops is limited, but there are occasional patients who develop extensively cross-reactive antibody responses (Zhang et al. (1999) J Virol 73, 5225-5230). One particular Env, designated R2 and derived from an HIV-1-infected individual, generated serum antibodies that exhibited extensive neutralizing cross-reactivity against many primary strains of HIV-1 of diverse virus subtypes (Dong et al. (2003) J Virol 77, 3119-3130; Zhang et al. (2002) J Virol 76, 644-655). R2 is also highly unusual as a naturally occurring HIV-1 Env in that it is be capable of mediating CD4-independent infection (U.S. Pat. No. 7,090,848; Zhang et al. (2002) J Virol 76, 644-655) Immunogenicity studies conducted in small animals and nonhuman primates have demonstrated that R2 induces neutralizing antibodies against multiple HIV-1 strains (Dong et al. (2003) J Virol 77, 3119-3130; Quinnan et al. (2005) J Virol 79, 3358-3369). The neutralizing cross-reactivity observed in those studies was greater than that previously reported in studies of other envelope immunogens (Labrijn et al. (2003) J Virol 77, 10557-10565).
These results were a major breakthrough, since they were the first demonstration that such a response was even possible. However, the potency of the response was modest, and not likely to be sufficient to result in durable immunity in a high proportion of vaccinated individuals. Therefore, research to identify methods to enhance the potency of the neutralizing response is much needed.
Preparation of soluble protein, which can be administered in a vaccine, that presents the same quaternary structure as the native trimeric complex on the surface of the virus is difficult, since extraction of the protein from the surface of the virus or cells is likely to substantially alter its quaternary structure.
The production of Env as gp140 is one approach that has been previously used (Zhang et al. (2007) Proc. Natl. Acad. Sci. U.S.A. 104:10193-10198; Dong et al. (2003). J. Virol. 77:3119-3130; Quinnan. et al. (2005) J. Virol. 79:3358-3369; Earl et al. (2001) J. Virol. 75:645-653). Under this approach, gp140 is produced in cell culture as a recombinant protein. The gp140 is a modified gp160, whereby the gp160 coding sequence is altered so that the protein produced lacks the amino acids necessary for protease cleavage, and further lacks the segments of gp41 that normally are imbedded in the viral membrane (transmembrane or TM segment) or in the interior of the virus or cell (cytoplasmic tail, CT). Since the gp140 protein is truncated so that the TM and CT segments are lacking, it is secreted by producing cells, and can be purified from tissue culture medium using non-denaturing conditions. Thus, the purified protein is at least partially in a trimeric form that presents a quaternary structure that is similar to that of the native protein on intact virus.
A prior study to assess antibody production was conducted in three groups of rabbits, with each group receiving different immunogens (Zhang et al. (2007) Proc. Natl. Acad. Sci. U.S.A. 104:10193-10198). One group received HIV-1 envelope glycoprotein R2gp120 in the adjuvant AS02A, a second group received the HIV-1 envelope glycoprotein R2gp140 in the adjuvant AS02A, and a third group received just the adjuvant AS02A.
Rabbits that received R2gp120 immunization developed a reasonably rapid and potent neutralizing antibody response that had very limited cross-reactivity, while the rabbits that received R2gp140 developed broadly cross-reactive neutralization that developed more slowly, and was lower in potency, as demonstrated in FIG. 1. One explanation for this observed difference is that neutralizing antibodies induced by R2gp120 with restricted cross-reactivity may be directed against high affinity, immunodominant, strain-specific epitopes, whereas those neutralizing antibodies induced by R2gp140 with broad cross-reactivity may be directed against lower affinity, cross-reactive epitopes.
As the R2gp140 recombinant preparation is a mixture of monomeric, trimeric, and multimeric proteins, the immunodominant, high-affinity epitopes on some of the protein species in the mixture override the development of an immune response against the important epitopes. Antibody responses typically induced by proteins tend to develop with kinetics more similar to the R2gp120-induced response than the cross-reactive response induced by R2gp140. That is, a potent response is obtained after a single immunization in adjuvant, and a potent booster effect is observed if a booster immunization is given a month or more later. In this respect, the R2gp120-induced response is typical, while the cross-reactive neutralizing response induced by R2gp140 is atypical. This difference indicates that B cells that elaborate the antibodies mediating highly cross-reactive neutralization are less effectively induced than the B cells that produce antibodies mediating neutralization with limited cross-reactivity.
Recent data further attests to the existence of potentially important differences in antigenic structure of R2gp120 and R2gp140. These data regard testing of sera from the same rabbits for the presence of antibodies binding to HIV-1 Env of different strains, and testing of ability of synthetic peptides to block neutralizing activity. The results of testing of immunoglobulin (Ig) binding to Env of different strains were also previously reported (Zhang et al. (2007) Proc. Natl. Acad. Sci. U.S.A. 104:10193-10198). Ig binding to the strain R2 Env used for immunization, and two other strains of Env was determined by enzyme-linked immunosorbent assay (ELISA), as shown in FIG. 2. The antibodies induced by gp120 immunization reacted much more with R2gp140 than with the gp140s from either of two other strains. In contrast, the antibodies induced by R2gp140 immunization reacted similarly with all three gp140s. The induction of strain-specific antibodies by immunization with gp120 is well established in the published literature.
The failure of R2gp140 immunization to induce such antibodies is unexpected, and indicates that the epitopes that induce the strain specific response are not effectively presented to the immune system by R2gp140 even though the full R2gp120 sequence is included in the protein. Strain-specific responses tend to be directed toward variable parts of the protein, rather than those sequences that are conserved among strains. Notably, the immunodominant variable region epitope in R2gp120 is variable region 3 (V3). The intriguing possibility is that the conformation assumed by R2gp140 is such that the immunodominant, variable region 3 epitope may be masked so that it cannot be seen by the immune system. This possibility is consistent with the evidence that demonstrates that a major reason why anti-V3 antibodies display limited neutralizing cross-reactivity is that the conformation of Env on the surface of the virus masks access to the critical region of the V3 loop.
Prior studies were conducted to determine whether antibodies directed against the V1, V2, or V3 regions of Env contributed to the neutralizing response induced by R2gp120 or R2gp140. For these studies peptides were synthesized that corresponded to sequences of these regions. This approach was taken because previous studies had demonstrated that the neutralization by antibodies directed against these regions could be blocked by the presence of soluble peptides. V1 and V2 region peptides had no effect on neutralization by the sera from either the R2gp120 or R2gp140 immunized rabbits. Similarly, V1 and V2 region deletion mutants were as susceptible to neutralization by the rabbit sera as virus presenting wild-type Env. In contrast, synthetic peptide homologous to the V3 region of R2 Env significantly blocked neutralization by sera from the gp120, but not gp140-immunized rabbits, as shown in FIG. 3. The results indicate that neutralizing antibodies directed against V3 contribute to the neutralizing response to R2gp120 but not the highly cross-reactive response induced by R2gp140. These results are consistent with the indication that V3 masking occurs in R2gp140.
Competition between high and low affinity epitopes for induction of B cell responses is well documented in the immunology literature. In fact, the successful induction of antigen-specific responses depends upon the amplification of responses resulting from high affinity interaction of B cells with antigen through the recruitment of T helper cells, and the deletion of B cell subsets that interact weakly with the antigen through apoptosis. In this manner, only antibodies with high affinity that are unlikely to cross-react with other, unrelated antigens are induced.
Epitopes that are immunodominant are those that are more successful in the competitive environment of B cell response induction. The observation that antibodies that mediate neutralization with restricted cross-reactivity develop as more typical responses, and that antibodies that mediate broad cross-reactivity develop more slowly and with lower potency indicates that the epitopes inducing the former are immundominant. Undoubtedly, there are dominant and non-dominant epitopes on R2gp140, and the sequences that form the dominant epitopes on R2gp120 are all present on R2gp140. However, there is a reasonable basis to hypothesize that these dominant epitopes are not presented to B cells effectively by conformationally intact trimeric Env.
Compelling evidence exists that antibodies that bind conformationally intact Env neutralize primary viruses cross-reactively. In contrast, antibodies that do not bind conformationally intact Env of any particular virus do not neutralize that virus. Conversely, an Env that cannot be recognized by an antibody molecule would not be competent to induce antibody production by a B cell expressing that molecule on its surface.
The comparative features of the immune responses induced by R2gp120 and R2gp140 indicate that the response induced by R2gp140 represents the additive effects of forms of the protein that resemble R2gp120 in immunogenicity and forms that are distinct from gp120 in that they present epitopes associated with highly cross-reactive neutralization. Further, the data indicate that the immunodominant epitopes may be effectively presented only by the gp120-like species, while the epitopes associated with broad neutralizing cross-reactivity are only presented on the oligomeric species. Thus, there is a need for an immunogen that is highly purified oligomer to selectively induce the highly cross-reactive neutralizing response. Such a purified oligomer is also of use to allow for the absence of competition from more dominant epitopes. A need exists for a purified oligomer to permit conversion of the cross-reactive response into a high potency, rapid antibody response.